The present invention relates to esters of aromatic sulfonic acids and the use of such acids in the inhibition of proteolytic enzymes. More particularly, the present invention relates to esters of aromatic sulfonic acids containing an amidino group and the use of such esters in the inhibition and inactivation of enzymes such as thrombin.
In the study of enzyme behavior, affinity labeling has provided an approach to identifying functional groups in enzymes that has had wide applicability. The success of this method depends on devising reagents which are substrate-like in structure, leading to an enzyme-reagent complex with subsequent covalent bond formation. The newly formed bond must be stable enough to permit structural study by standard methods of protein chemistry.
It has been appreciated that the same approach might be of value in arriving at therapeutically useful agents since inactivation with covalent bond formation would confer maximal effectiveness to inhibitors whose selectively arises from substrate-like structural features.
With specific regard to the enzyme, thrombin, this is a proteolytic enzyme which catalyzes the conversion of fibrinogen to fibrin and thus is essential in the clotting mechanism of blood. It is present in the blood in the form of prothrombin under normal conditions; when bleeding begins, the prothrombin is converted to thrombin, which in turn activates the formation of fibrin. Thus, the inactivation of thrombin would result in an anticoagulant action which would be useful in various therapeutic treatments and studies.
With the goal of obtaining a reagent capable of discriminating among trypsin-like enzymes and inactivating one of them, nitrophenyl m- and p-amidinophenylmethanesulfonate have been synthesized, in accordance with the present invention, such compounds having specific use as potential affinity labels. Both compounds were found to be competitive inhibitors of trypsin, thrombin, plasmin, chymotrypsin, and plasma kallikrein. However, in the case of thrombin alone, the p-isomer achieves a covalent modification irreversibly inactivating the enzyme. The results obtained, as discussed hereinafter, demonstrate the selectivity possible with such an active site-directed reagent even among enzymes with homologous active centers.
A number of physiologically important proteolytic enzymes appear to be derived from trypsin by gene duplication. Several of these which are liberated in plasma by zymogen activation such as thrombin, plasmin, and kallikrein have key roles in coagulation, fibrinolysis, and permeability. The ability to selectively inactivate one of these or other trypsin-like protease would be of therapeutic value. Since these enzymes have the hydrolytic mechanism common to serine proteases and a specificity site for a basic amino acid side chain, it seemed likely that their physiological selectivity in proteolysis would depend on recognition of larger regions of their normal macromolecular substrates. Correspondingly, it seemed unlikely that small reagents comparable in size to a single basic amino acid could selectively inhibit one of these trypsin-like enzymes and not the others.
On the other hand, it has been observed that esters of p-guanidinobenzoic acid, an analog of lysine and arginine, form relatively stable acyl-enzyme derivatives with trypsin-like enzymes. Since thrombin and plasmin differ in the duration of their inactivation as p-guanidinobenzoyl enzymes, it appeared that there are kinetic differences between thrombin and plasmin that may be exploited for selective inactivation by this type of quasi-substrate. In general, the results supported the earlier impression that the immediate region of the hydrolytic center of thrombin is more flexible than that of plasmin, i.e., less likely to adopt an acyl-enzyme configuration which excludes the productive approach of a water molecule essential for deacylation. The latter situation appears to explain the stability of one acyl-enzyme, indoleacryloyl-chymotrypsin, and is considered as probably applying to other relatively rigid acyl residues. Since an adequately stable acyl-thrombin was not found, attention was turned to the possibility of selective formation of a sulfonyl thrombin derivative by affinity labeling since prior experience indicates that this type of serine protease derivative is most stable to hydrolysis. However, in such a case, selectivity among trypsin homologs would have to be achieved by differences in sulfonylation rather than in desulfonylation.
The most favorable result would therefore be a qualitative difference in which the reagent, although complexing with a variety of trypsin-like enzymes as expected in the affinity labeling approach, would achieve covalent modification with only one member of the group, presumably due to small differences in geometry in the region of the active center that provide the correct orientation for sulfonylation of the active center only with a single member of the group.
In accordance with the present invention, nitrophenyl p- and m-amidinophenylmethanesulfonate, hereinafter sometimes referred to as the p- and m-isomers, have been prepared. These compounds have been found to be effective inhibitors of thrombin, with the p-isomer providing specific inactivation of this enzyme.